Trimetrexate, 2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl] quinazoline is a useful pharmaceutical compound known to have antineoplastic, antiparasitic and antibacterial activity. The trimetrexate free base has the structure: ##STR1##
Trimetrexate is an inhibitor of the enzyme dihydrofolate reductase (DHFR) that is known to catalyze the synthesis of nucleotide precursors of DNA and RNA. Trimetrexate glucuronate is categorized as a folate antagonist, and has been clinically evaluated and is now approved for use in the treatment of Pneumocystis carinii pneumonia (PCP) in patients with acquired immune deficiency syndrome (AIDS) (Physicians' Desk Reference, 51st ed. (1997)). Inhibition of the DHFR enzyme results in cellular depletion of reduced folates, which are necessary for various cellular processes, including RNA and DNA synthesis, and ultimately results in cell death. It is this property that gives trimetrexate its antineoplastic, antiparasitic and antibacterial activity.
Trimetrexate has demonstrated antitumor activity against a range of experimental murine and human tumor cell lines, in vitro and in vivo. For example, trimetrexate has shown antitumor activity against murine cell lines such as L1210, L5178Y, S-180, W-256 in vitro. In addition, trimetrexate has shown antitumor activity against human tumor cells lines derived from breast, colon, lung, ovary, renal and melanoma cells in vitro. In vivo studies have demonstrated that trimetrexate has therapeutic utility against murine tumors such as B16 melanoma, colon 26 and 38, L1210 and p388 leukemia and CD8F mammary tumors. Other possible uses for trimetrexate include the treatment of malaria, psoriasis, rheumatoid arthritis and prophylaxis against Pneumocystis carinii pneumonia.
Trimetrexate as the free base is lipophilic, with very low water solubility (&lt;0.1 mg/mL). Various trimetrexate salts with greater aqueous solubility are known. U.S. Pat. No. 4,376,858 to Colbry ("Colbry") discloses trimetrexate glucuronate as a preferred salt, due to its superior water solubility (&gt;50 mg/mL), stability and the low toxicity of glucuronic acid. Colbry additionally discloses a method of preparing trimetrexate glucuronate wherein trimetrexate and glucuronic acid are dissolved in warm methanol and ethyl acetate and the solution is then cooled to precipitate the glucuronate salt. Additional trimetrexate salts and methods for making them are described in PCT publication WO96/21451.
Hicks et al., J. Labelled Compounds Radiopharm., 29, 415 (1991), discloses another method of manufacture of trimetrexate glucuronate salt. In this method, trimetrexate and glucuronic acid are added to an aqueous solution in an ampoule, followed by lyophilization to form a solid, amorphous salt.
Trimetrexate is available as a commercial drug product under the commercial name Neutrexin.RTM. (U.S. Biosciences). The drug product was developed by Warner-Lambert/Parke-Davis as an injectable formulation presented as a 5 cc flint glass vial (USP Type I) containing 25 mg of trimetrexate and 15.35 mg D-glucuronic acid. The glucuronic acid is present in the formulation to help solubilize trimetrexate which is intrinsically insoluble in water. Neutrexin.RTM. provides trimetrexate glucuronate as a lyophilized powder, and is reconstituted prior to use in conjunction with leucovorin for the treatment of moderate to severe Pneumocystis carinii pneumonia in immunocompromised patients, e.g., those suffering from AIDS (U.S. Bioscience's Neutrexin for PCP, Scripp 1886/87, 31 (1994)).
The trimetrexate free base of the prior art, hereinafter referred to as "trimetrexate", is not stable for long-term storage, and rapidly degrades. To overcome the storage stability problem, trimetrexate is typically stored as a salt, due to the increased stability found for some trimetrexate salts. Stetson et al., J. Chromatography, 464, 163-171 (1989), discusses the stability of the trimetrexate glucuronate salt obtained from Warner Lambert/Parke-Davis, Pharmaceutical Research Division. This salt is apparently that obtained by the process disclosed in U.S. Pat. No. 4,376,858. Stetson indicates that the glucuronate salt has a half-life in solution of 51.6.+-.0.8 days at 37.degree. C.
The instability of trimetrexate creates numerous disadvantages. It is convenient to manufacture bulk trimetrexate and the final pharmaceutical formulation in different facilities to take advantage of specialization at different manufacturing plants, and economies of production, shipping, packaging, storage and the like. However, the instability of the prior art trimetrexate makes achieving these goals difficult. Additionally, degradation of trimetrexate between production and final formulation disadvantageously decreases the drug yield, and introduces additional costs in the manufacturing process necessary to produce a pharmaceutical-grade product.
Despite the disadvantages of trimetrexate, other, more stable non-salt forms of trimetrexate useful in pharmaceutical applications have not been identified. Indeed, USAN and the USP Dictionary of Drug Names show trimetrexate in only two forms: the anhydrous free base (trimetrexate), and the trimetrexate glucuronate salt.
Hempel et al., Cancer Biochem. Biophys., 10, 25-30 (1988) ("Hempel"), discloses molecular structures of crystalline trimetrexate acetate monohydrate. Hempel additionally identifies a poorly-characterized, poly-hydrated trimetrexate free base. In Hempel, trimetrexate polyhydrate was crystallized from a methanol/water DMSO solution, and characterized by x-ray diffraction. The trimetrexate polyhydrate crystals belong to the C2/c space group and have structural parameters of a=36.051 .ANG., b=11.765 .ANG., c=10.623 .ANG. and .beta.=105.69.degree.. The polyhydrate obtained by Hempel, however, is poorly characterized, having an unknown hydration number and consisting of poor quality crystals containing a number of disordered water molecules. In addition, Hempel does not disclose any stability advantages of the polyhydrated trimetrexate form.
Sutton et al., J. Med. Chem., 30, 1843-48 (1987) ("Sutton"), reports the crystal structure of a DMSO-H.sub.2 O adduct of trimetrexate, trimetrexate dimethyl sulfoxide hydrate. Trimetrexate was crystallized as the DMSO hydrate, and the crystals analyzed by x-ray diffraction. The crystals of the trimetrexate-DMSO-H.sub.2 O adduct were found to be triclinic, belonging to the P-1 space group, and having structural parameters of a=9.423 .ANG., b=11.180 .ANG., c=12.399 .ANG. and .beta.=75.10.degree.. However, the only form of trimetrexate produced and analyzed was the DMSO-H.sub.2 O adduct. In addition, Sutton does not disclose any thermal stability advantages of the DMSO-H.sub.2 O adduct. A stable, non-salt form of trimetrexate has not previously been produced and characterized.
It should be clear from the above that there is a need for a stable trimetrexate bulk drug substance that can be used to more efficiently and more cost-effectively produce a final trimetrexate pharmaceutical product. Further, there is a need for an efficient and rapid synthesis for this stable bulk drug substance.